Muscle wasting occurs during aging, HIV infection, cancer, and numerous other pathological conditions, resulting in a significant decrease in quality of life and a financial burden of $18.5 billion in 2000. While 3D in vitro models of skin and lung tissue have proven essential in elucidating mechanisms of homeostasis and disease progression, analogous models of skeletal muscle do not exist. We propose a 3D model of primary human skeletal muscle that utilizes an engineered extracellular matrix (eECM), gradients of chemotactic cues, and cellular patterning. This collaborative proposal combines complementary expertise in cell microenvironment engineering and human muscle progenitor cell (hMuPC) and myoblast biology. Aim 1 is to develop and optimize a 3D eECM to enhance the proliferation of hMuPCs. Previous results show that hMuPCs are critically responsive to the biochemistry and biomechanics of the microenvironment and have diminished proliferation and regeneration following 2D culture. Customized eECM will be made from a protein-engineered biomaterial that enables independent tuning of biomechanics (elastic moduli = 1-100 kPa) and cell-ligand density (0-100,000 ligands/micron3). Viability, proliferation, and myogenic differentiation of hMuPCs will be directly compared between 2D and 3D cultures utilizing identical eECM. Aim 2 is to develop a 3D in vitro model of hMuPC migration. Little is known about the soluble cues that regulate hMuPC migration to sites of regeneration in vivo. Time-lapse imaging of hMuPC migration speed, directional persistence, and filopodia extension will be performed in a microfluidic device that enables the formation of stable concentration profiles. Migration will be compared on 2D and in 3D eECM in response to gradients and uniform concentrations of putative chemotactic cues. Migration in response to cell lysates from young (18-25 years old), old (60-80 years old), and dystrophic human skeletal muscle biopsies will be quantified to identify potential novel regulators of chemotaxis. Aim 3 is to develop a 3D patterned mimic of human skeletal muscle tissue. Human myoblasts will be cultured on patterned eECM to induce myotube fusion and alignment. Fiber fusion rate, maturity, nuclear index, and alignment will be compared on eECM of varying pattern geometry, biomechanics, and biochemistry. Multiple sheets of aligned myotubes will be layered together with hMuPCs to create a dynamic model of regenerating muscle tissue. These aims will lead to new 3D technologies for tissue culture, fundamental new insights in skeletal muscle biology, and potential new clinical therapies to activate hMuPCs and stimulate regeneration of muscle damaged during wasting and aging. (End of Abstract)